Leonard C K, Spellman M W, Riddle L, Harris R J, Thomas J N, Gregory T J
Department of Medicinal Chemistry, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1990 Jun 25;265(18):10373-82.
This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.
本报告描述了在中国仓鼠卵巢细胞中表达产生的1型人类免疫缺陷病毒重组包膜糖蛋白（rgp120）的结构特征。采用rgp120的酶切和反相高效液相色谱法来确认该蛋白的一级结构、确定链内二硫键，并表征N-糖基化的潜在位点。鉴定出的所有胰蛋白酶肽段均与从cDNA序列预测的一级结构一致。胰蛋白酶图谱研究结合用金黄色葡萄球菌V8蛋白酶或肽：N-糖苷酶F处理分离的肽段，随后用天冬氨酸内肽酶Asp-N处理，从而确定了rgp120的所有九个链内二硫键。通过测定连接的碳水化合物结构对肽：N-糖苷酶F和内切β-N-乙酰氨基葡糖苷酶H的敏感性，对24个潜在的N-糖基化位点进行了表征。将酶促去糖基化的rgp120的胰蛋白酶图谱与单独处理的肽段的埃德曼降解和快原子轰击质谱联用，以确定这些位点中哪些被糖基化以及存在何种类型的结构。结果表明，gp120的所有24个位点均被利用，其中13个位点以复合型寡糖为主要结构，11个位点主要含有高甘露糖型和/或杂合型寡糖结构。
